The present invention relates generally to the field of immunology and, more particularly, to antigen-xcex12-macroglobulin complexes, the facile and reproducible preparation of antigen-xcex12-macroglobulin complexes, and their subsequent uses, including the enhancement of host immunocompetence and the preparation and administration of vaccines for prevention and treatment of disease states.
In general, antigens are xe2x80x9cpresentedxe2x80x9d to the immune system by antigen presenting cells (APCs), including, for instance, macrophages, dendritic cells and B-cells in the context of major histocompatibility complex molecules (MHCs) which are present on the APC surface. Normally, natural antigens and molecules supplied as immunogens are thought to be taken up and partially digested by the APCs, so that smaller pieces of the original antigen are then expressed on the cell surface in the context of MHC molecules.
It is also presently understood that T-lymphocytes, in contrast to B-lymphocytes, are relatively unable to interact with soluble antigen. Typically T-lymphocytes require antigen to be processed and then expressed on the cell surface of APCs in the context of MHC molecules as noted above. Thus, T-cells, and more particularly, the so called xe2x80x9cT-cell receptors,xe2x80x9d are able to recognize the antigen in the form of a bimolecular ligand composed of the processed antigen and one or more MHC molecules. In addition to presenting antigens on MHC molecules, the APC must be activated to express co-stimulatory molecules, such as B7/B1, before effective stimulation of T-cells can occur.
Many epitopes on proteins, including both foreign and endogenous proteins, are generally unrecognized or only weakly recognized by the immune system. These epitopes therefore elicit little or no antibody or other immune response, or at most, only a weak response. It has therefore been difficult, and in some instances, impossible to raise antibodies against such epitopes. In contrast, other epitopes elicit extraordinarily strong immune responses, in some instances, to the exclusion (or partial exclusion) of other epitopes within the same antigen molecule. Such epitopes can be termed xe2x80x9cimmunodominant.xe2x80x9d
A separate problem arises in the preparation and administration of vaccines, and particularly vaccines that present peptide antigens. Traditional methods for preparing such vaccines that present antigens as macromolecules through conjugation to protein carriers or polymerization are often unable to induce cytotoxic T lymphocytes (CTL) response in vivo. In such instances an adjuvant is usually added. Use of an adjuvant in the immunizing protocol has the advantage of enhancing the humoral response but has mixed results in priming specific CTL response. Unfortunately, popular adjuvants used in laboratory animals, such as Freund""s complete adjuvant, are too toxic and unacceptable for humans. Ideally, protection against viral infection is best provided by both humoral and cell-mediated immunities, including long-term memory and cytotoxic T cells.
For example, the human immunodeficiency virus (HIV), the etiologic agent most closely associated with the acquired immunodeficiency syndrome (AIDS), has become an important objective for various vaccine developments. The predominant vaccine strategy has focused on the use of the envelope protein antigens gp120 and gp160 of HIV-1 produced by recombinant DNA technology. However, the full promise of their use in vaccines cannot presently be realized unless they are administered along with an effective adjuvant.
The targeting of antigen (abbreviated Ag) to APC has been extensively studied in vitro and in vivo [For review see (1, 2)]. Techniques that have been used include encapsulating Ag into liposomes (3, 4), crosslinking Ag to antibodies directed against surface proteins (5-9), and forming immune complexes for recognition by FcR (10). A complementary approach of decorating B cell surfaces with mAb recognizing a particular Ag also conferred enhanced ability to present that Ag (11). The capacity for Ag uptake by different APC appears to correlate with efficiency of presentation (12), although Ag focusing or intracellular signaling may also contribute. In general, targeting of Ag to the APC surfaces appears to enhance the immune response.
While B-cells possess specific receptors, surface Ig, for capturing the Ag they present efficiently (13,14), macrophages and other non-B cell APCs must utilize other mechanisms. These may include phagocytosis of particulate or cellular Ag and enhanced endocytosis of opsonized Ag or immune complexes. Yet, the efficient uptake and presentation of soluble Ag by these non-B cell APCs in naive animals is not fully understood. A receptor-mediated process might be involved.
Among the APCs, the macrophages are of particular interest by virtue of the central role that they play in the regulation of the activities of other cells of the immune system. Macrophages act as effector cells in microbial and tumor cell killing as well, and are believed to secrete numerous cytokines that orchestrate many of the diverse aspects of the immune response. The ability of macrophage to regulate a range of immunologic events is in part a function of their expression of Ia surface antigens. The expression of membrane Ia antigens is essential for the induction of specific T cell responses to antigens (15).
The effective internalization and processing of diverse proteins forms a central issue in antigen presentation by macrophages. The immune system must balance the capacity for interacting with vast numbers of dissimilar molecules with the requirements for efficiently responding to very low amounts of Ag. Although macrophages are able to sample their environments through pinocytosis, a need for more efficient means of internalization, such as a receptor-mediated system, has been suggested (16). The targeting of Ag to surface receptors on macrophages or B-cells, either by artificial crosslinking or by exploiting membrane Ig, enhances the efficiency of presentation (1,16,17); however, a naturally occurring antigen presentation system in macrophages has not yet been identified.
The xcex1-macroglobulins and the complement components C3, C4, and C5 comprise a superfamily of structurally related proteins. The xcex1-macroglobulin family includes proteinase-binding globulins of both xcex11 and xcex12 mobilities. The most extensively studied xcex1-macroglobulin is human xcex12-macroglobulin (xcex12M), a large tetrameric protein capable of covalently binding other proteins (19-27) and targeting them to cells bearing the xcex12M receptor (27-30). Although size and charge may affect the extent of binding, xcex12M can incorporate proteins bearing nucleophilic amino acid side chains in a relatively non-selective manner. This rapid covalent linking reaction is restricted, however, to a window of time initiated by proteinase-induced conformational change, during which an internal thioester on each subunit becomes susceptible to nucleophilic substitution (20,21,31). Thus, xcex12M, C3 and C4 are evolutionarily-related thioester-containing proteins that undergo conformational and functional changes upon limited proteolysis (32,33), resulting in possible formation of thioester-mediated covalent bonds with targets such as proteinases, cell-surface carbohydrates or immune complexes, respectively.
Human xcex12-macroglobulin (xcex12M) is an abundant protein in plasma (2-5 mg/ml). It consists of four identical subunits arranged to form a double-sided molecular xe2x80x9ctrapxe2x80x9d (34). This trap is sprung when proteolytic cleavage within a highly susceptible stretch of amino acids, the xe2x80x9cbait region,xe2x80x9d initiates an electrophoretically detectable conformational change that entraps the proteinase (35). The resulting receptor-recognized xcex12M is efficiently internalized by macrophages, dendritic cells, and other cells that express xcex12M receptors [reviewed in (36); see also (37)], one of which has recently been cloned and sequenced (38, 39). Reaction of xcex12M with methylamine results in a similar conformational change to a receptor-recognized form of xcex12M. Methylamine-treated and proteinase-treated xcex12M are equivalent with regard to binding, internalization and signaling. Amine-treated or protease-treated xcex12-macroglobulin is termed xcex12-macroglobulin* and abbreviated xcex12M*. Receptor-recognized xcex1-macroglobulins from different animal species cross-react with similar affinities for the xcex12M receptor regardless of the proteinase used [See (36,40,41) for review]. The additional binding of non-proteolytic proteins does not appear to affect the rate of internalization even when artificial crosslinking is employed (28,29,42). Therefore, regardless of the mechanism of binding, proteins complexed with xcex12M* can be effectively internalized.
The possible role of xcex12-macroglobulin as a delivery vehicle for antigens, hormones or enzymes has been reviewed previously in the art (43-47). In the past, there have been numerous other studies suggesting a role for xcex12M in immune modulation (Reviewed in (48)).
As described above and in the cited literature, antigens which are not themselves proteinases are unable to become covalently bound to xcex12-macroglobulin by co-incubation of the antigen with xcex12-macroglobulin. Covalent incorporation of a potential antigen into the xcex12-macroglobulin molecule requires the participation of a proteolytic enzyme to cleave the xcex12-macroglobulin molecule as a necessary precursory step to then permit its thiol ester to react with and thus bind the antigen. While the use of a proteolytic enzyme allows the in-vitro preparation of the desired antigen-xcex12-macroglobulin complex, the requirement for a proteolytic enzyme in this process is significantly deleterious to the structural and epitopic integrity of the antigen desired to be complexed with xcex12-macroglobulin, as it may be proteolyzed into smaller fragments during the preparation of the complex or after it has bound to the xcex12-macroglobulin. Furthermore, the proteolytic enzyme itself is always incorporated into the complex, thus imposing steric hindrance limiting the size of the antigen that is incorporated into xcex12M to about 40 kilodaltons. Thus, the facile and reproducible preparation of a complex between xcex12-macroglobulin and an antigen of any size for the purpose of, for example, using the complex as a vaccine, is not straightforward. The structure of the antigen may be materially altered by proteolytic cleavage, and the extent and purity of antigen and other components incorporated into the xcex12-macroglobulin may affect the quality and quantity of final complex formed.
Other means for preparing antigen-xcex12-macroglobulin complexes are also not straightforward. Treatment of xcex12-macroglobulin with a low molecular weight amine (nucleophile) to cleave the thiol ester achieves the conversion to the desired receptor-recognized form of xcex12-macroglobulin; however, the amine-modified thiol ester is no longer able to bind antigen at the glutamyl residue of the thioester. Several investigators have evaluated whether amine-treated (e.g., methylamine-treated) xcex12-macroglobulin has the capability of binding an antigen, including proteinases. No covalent linking of trypsin or elastase was seen when methylamine-treated xcex12M was incubated with these enzymes for several hours at 23xc2x0 C. (49, 50). Thus, preparation of a covalent antigen-xcex12M* complex in the absence of proteinase was heretofore unachievable.
A need therefore exists for the development of a simple and reproducible method for the preparation of a covalent complex between xcex12-macroglobulin and a desired antigen without limitation to size, avoiding the use of proteolytic enzymes and reproducibly providing a vaccine or other material in which the antigen is stable and structurally defined for use in modulating the immune response. It is towards these goals that the present invention is directed.
The invention described herein relates generally to the modulation of the immune response by a structurally-defined and stable antigen covalently coupled to the receptor-recognized form of xcex12-macroglobulin (xcex12M*). The antigen-xcex12-macroglobulin complex of the present invention comprises a covalent adduct of the antigen and xcex12-macroglobulin with an intact bait region, the antigen incorporated into the amine-activated form of xcex12-macroglobulin by nucleophilic exchange in the absence of proteolytic enzymes. The antigen may be covalently bound to the glutamyl or cysteinyl residues of the cleaved thiol ester of the xcex12-macroglobulin molecule, or it may be bound to both. One or more antigens may be bound to the complex. More particularly, the present invention is directed towards facile and reproducible methods of preparing the covalent complex between the antigen and the receptor-recognized form of xcex12-macroglobulin in which conditions for the preparation of the complex do not compromise the integrity of the antigen. The complex prepared by the procedures described herein provide a stable and defined material for use as a vaccine or other reagent for modulating immunocompetence in an animal or in an in vitro system. The size of the coupled antigen is not limited. Furthermore, the complexes described herein may be used for increasing the immune response to an otherwise poorly immunogenic antigen, and, under certain conditions, for the suppression of the immune response to a particular antigen.
In contrast to the prior art antigen-xcex12-macroglobulin* complexes, and procedures for preparing such complexes, whereby coupling is achieved by the concomitant use of a proteolytic enzyme to cleave xcex12-macroglobulin and to render the thiol ester available for reaction with an antigen, in the practice of the present invention, the antigen is coupled to a previously nucleophile-activated xcex12-macroglobulin, in the absence of proteolytic enzymes, using an elevated temperature and correspondingly-appropriate duration of incubation to achieve the desired coupling. Thus, the xcex12-macroglobulin in the complex of the present invention has an intact bait region. xcex12-Macroglobulin first may be activated by a low molecular weight amine such as ammonia, methylamine, ethylamine, propylamine and the like. Ammonia and methylamine are preferred. The antigen may be incubated with the amine-activated xcex12-macroglobulin at a temperature of from about 35 C. to 55 C., and for an appropriate duration to achieve the desired coupling. Selection of the appropriate temperature may be made depending on the stability of the particular antigen. For example, at 50xc2x0 C., coupling may be achieved in 1-5 hour; at 37xc2x0 C., the coupling may be achieved at 24 hours. Preferred conditions for an antigen stable at 50xc2x0 C. is 1-5 hours. Preferred conditions for an antigen stable at 37xc2x0 C. is 24 hours.
The xcex12-macroglobulin used in the present invention be native protein or that produced recombinantly, using well known techniques in molecular biology.
Suitable antigens for coupling to xcex12-macroglobulin to prepare the complexes of the present invention include nucleophiles, and extend to and include peptides, proteins, carbohydrates, cytokines, growth factors, hormones, enzymes, toxins, nucleic acids such as anti-sense RNA, as well as other drugs or oligonucleotides.
In a further embodiment, the antigen may be mildly oxidized, for example, by N-chlorobenzenesulfonamide, to increase the amount of antigen coupled to xcex12-macroglobulin by the methods of the present invention.
The complex formed by the procedure of the present invention may be introduced to a cell culture system or host animal, or to a target tissue or organ, where it is believed that xcex12M* augments presentation of the desired antigen and the development of the corresponding immune response will occur.
One of the advantages of the present invention and a particular feature thereof, resides in the fact that the complex prepared by the covalent binding of xcex12M to a given antigen by the procedures described herein, can be administered as a vaccine without need for an adjuvant. In view of the difficulties that are experienced when adjuvant formulations are included in vaccines, the preparation of vaccines in accordance with the present invention represents a significant improvement and offers the promise of a far more efficient vehicle for antigen presentation, and one which will avoid many of the drawbacks such as toxicity and the like that are experienced with current adjuvant-containing formulations.
Also, the complexes of the present invention have particular utility in their affinity for macrophages, and other cells that bind or internalize xcex12M. The scope of antigens, immunogens or immune modulating molecules that may be associated in the complex of the present invention is equally diverse, as it extends from oligonucleotides, proteins, peptides, cytokines, toxins, enzymes, growth factors, antisense RNA and drugs, to other carbohydrates that may exhibit some desired modulatory effect on the target cells. There is a need only for a nucleophilic group, such as an amine, sulfhydryl, or hydroxyl, to exchange with the amine present on xcex12-macroglobulin*. The invention is therefore contemplated to extend to these variations within its spirit and scope.
A further advantage of the invention is that it provides for independently targeting a receptor-binding xcex12M, as well as complexes of the invention comprising these components, for endocytosis or for cell signaling and activation. Proper activation of the APC is necessary for effective antigen presentation and effective stimulation of the immune response in general.
It is contemplated that both positive and negative regulation of the antigenicity of epitopes can be achieved. For example, by rendering epitopes recognized, or recognizable, antibodies can be raised to recognize and bind to the antigen. Enhanced antigenicity and the ability to raise antibodies to otherwise weak, scarce or ineffective epitopes finds great utility not only, for example, in vaccine applications in animals, including humans, but also in producing antibodies which can be used as reagents for, among other uses, binding, identifying, characterizing and precipitating epitopes and antigens, such as the production of antibodies against scarce antigens for research purposes. Preferably, the immunogenicity of a given antigen is enhanced according to the methods of the invention.
Alternatively, this invention contemplates the down regulation or suppression of immune responses to immunodominant epitopes, by the preferential stimulation of immune responses to otherwise xe2x80x9csubordinatexe2x80x9d epitopes, or by the introduction of agents or factors that on presentation, would selectively suppress the immunogenicity of the target epitope. This additional ability to modulate antigenicity may be useful, for example, in immunizing animals, including humans, and also in producing antibodies which are reactive towards otherwise silent or weakly antigenic epitopes. Such antibodies are also useful for, among other things, binding, identifying, characterizing and precipitating epitopes and antigens in vivo and in vitro.
The invention described herein also preferably includes the antibodies produced by the methods described herein or in response to the immunogens, prepared as described herein, said antibodies including monoclonal, polyclonal and chimeric antibodies, as well as immortal strains of cells which produce such antibodies, for example hybridomas which produce monoclonal antibodies which recognize the molecules and other antigens of interest. Advantageously, such antibodies can be prepared against epitopes on the antigen that are normally secondary or even suppressed.
The invention also encompasses cellular immune system components, e.g., T-lymphocytes raised in response to such antigens or immunogens, pharmaceutical compositions containing the antigens, antibodies or cellular immune system components and various methods of use.
The invention provides for enhancing the efficiency of immunizations. This can have useful application not only for potential therapeutic interventions, in particular vaccinations, but also for production of antibodies or primed lymphocytes (T or B) against scarce antigens for research purposes.
Accordingly, it is a principal object of the present invention to provide a structurally defined and stable complex of an antigen with xcex12-macroglobulin for the purposes described herein.
It is another object of the invention to provide a stable complex comprising one or more intact biomolecules and activated xcex12-macroglobulin, in which each of the biomolecules is covalently bound to an amino acid residue of the cleaved thiol ester of xcex12-macroglobulin. The biomolecule may be bound to the glutamyl residue, or to the cysteinyl residue, or to both residues. The biomolecule may be a peptide, protein, carbohydrate, cytokine, growth factor, hormone, enzyme, toxin, anti-sense RNA, a therapeutic drug, an oligonucleotide, lipid, DNA, an antigen, an immunogen, or an allergens. The biomolecule may have a molecular weight of between about 0.5 and 100 kilodaltons.
It is another object of the invention to provide an immunogen that comprises an antigenic molecule having at least one epitope in a complex with xcex12-macroglobulin. The immunogen is a complex prepared by the sequential steps of activating xcex12-macroglobulin by incubation with a nucleophilic compound to form nucleophile-activated xcex12-macroglobulin, removing the excess nucleophilic compounds, and incubating the nucleophile-activated xcex12-macroglobulin with the biomolecule.
It is yet another object of the present invention to provide a method for the preparation of a covalent complex between one or more intact biomolecules and xcex12-macroglobulin by carrying out the steps of 1) activating said xcex12-macroglobulin by incubation with a nucleophilic compound to form nucleophile-activated xcex12-macroglobulin; 2) removing excess nucleophilic compound; and 3) incubating the nucleophile-activated xcex12-macroglobulin with said biomolecule.
It is yet a further object of the present invention to provide an immunogen which comprises an antigenic molecule in a complex with xcex12-macroglobulin, wherein the antigenic molecule has at least one epitope, and in which the xcex12-macroglobulin is capable of binding a receptor for xcex12-macroglobulin. In another embodiment, a method of rendering a poorly immunogenic epitope on an antigen recognizable by the immune system by preparing a complex between reacting said antigen molecule with xcex12-macroglobulin, exposing an antigen presenting cell having major histocompatibility complex to the complex; and contacting said antigen presenting cell with lymphocytes.
It is still a further object of the present invention to provide a vaccine which comprises an antigen-xcex12-macroglobulin complex prepared by the methods herein. In a further embodiment, a method of producing T-lymphocytes which recognize an antigen is described which comprises administering to a mammal a T-lymphocyte priming effective amount of a complex comprising an antigen and xcex12-macroglobulin prepared in accordance with the present invention, which is capable of binding a receptor for xcex12-macroglobulin; and harvesting said T-lymphocytes from the mammal. In a still further embodiment, a method of treating or preventing an infectious disease, an autoimmune disease or cancer in a mammalian patient in need of such treatment or prevention is described, comprising administering to the patient an effective amount of an immunogen comprised of a complex comprising an antigen and xcex12-macroglobulin in accordance with the present invention, which xcex12-macroglobulin is capable of binding a receptor for xcex12-macroglobulin, in an amount effective for modifying the immune response to said antigen.
It is a further object of the present invention to provide a method for the preparation a structurally defined and stable complexes of particular antigens with xcex12-macroglobulin which may be carried out easily and reproducibly for the various uses herein.
It is a still further object of the present invention to provide a method for the preparation of corresponding complexes as aforesaid that facilitate improved immune recognition and activation.
It is a still further object of the present invention to provide a method and corresponding complexes as aforesaid that can be used to selectively activate epitopes in distinction to other immunodominant epitopes.
It is a still further object of the present invention to provide a method for the facile development of clinically significant amount of antibodies directed against scarce antigens.
Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description which proceeds with reference to the following illustrative drawings.